Elsevier

Talanta

Volume 129, 1 November 2014, Pages 529-538
Talanta

High-performance liquid chromatographic and mass spectrometric analysis of fluorescent carbon nanodots

https://doi.org/10.1016/j.talanta.2014.04.008Get rights and content

Highlights

  • A simple HPLC method is applied to separate a very complex mixture of carbon nanodots.

  • The elution order follows with the sizes of carbon nanodots from the smallest to the largest.

  • Dialysis membrane is used to remove the reaction matrix from the carbon nanodots product.

Abstract

Amino/hydroxyl-functionalized fluorescent carbon nanodots (C-NanoD) are conveniently synthesized based on hydrothermal carbonization of chitosan at 180 °C. Dialysis membranes with small cut-off masses (500–1000 Da) were found useful for removing the side-products and low molecular mass species to purify the C-NanoD product. Herein, reversed-phase high-performance liquid chromatography (RP-HPLC) has been successfully applied to fractionate the C-NanoD product. The elution order of the C-NanoD species present in the sample follows approximately their core sizes from small to large. The separated C-NanoD fractions are collected and characterized by UV absorption spectroscopy, photoluminescence (PL) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and transmission electron microscopy (TEM). All the C-NanoD fractions display a distinctive absorption band at 300 nm, attributing to the n→π⁎ transition of Cdouble bondO bond. The PL spectra of the fractions display emission peaks at 400–415 nm which are slightly red-shifted with their increase in relative molecular masses. The C-NanoD fractions are fully anatomized by MALDI-TOF MS, displaying their fragmentation mass ion features. The core sizes of some selected C-NanoD are determined as 1.6, 1.8, 2.5, and 3.1 nm by TEM which are in consistent with their HPLC elution order. The findings highlight the virtues of RP-HPLC to fractionate and reveal the unique characteristics of individual C-NanoD species present in an as-synthesized C-NanoD product which may have potential applications in the fields of bioanalysis, bioimaging, catalysis, chemosensing, energy storage, and optoelectronics device.

Introduction

In recent years there is a huge interest in carbon nanodots (C-NanoD) primarily because of their unique properties in chemical inertness, good stability without photo-bleaching and optically non-blinking [1], [2] size- and wavelength-dependent luminescence emission [2], ease of synthesis and excellent biocompatibility [3], [4], low toxicity [5], cost-effective raw materials [6], promising up-conversion property [7], [8], and environmental friendliness [9] in comparison to conventional semiconductor quantum dots (QDs). Small C-NanoD are one of the latest rising stars among the carbon nanomaterial family, which upon surface functionalization by organic molecules were found to exhibit bright and colorful fluorescence emissions [10]. Most notably, they have the potential to replace the currently used toxic semiconductor-based QDs. However, this fluorescent C-NanoD is not well studied as compared to other carbon nanomaterials such as carbon nanotubes, fullerenes and graphenes. The understanding of the origin of fluorescence in C-NanoD is still under investigation. Sun et al. [10] and Wang et al. [11] proposed that the fluorescence emission in C-NanoD is attributed to radiative recombination of the photo-induced electron and hole that is confined to the carbon nanoparticle (NP) surface. This resembling phenomenon is found in nanoscale semiconductors [12]. Baker and Colón [13] suggested that the surface groups have strong influence on the fluorescence emission of C-NanoD which is derived from the surface energy trap state. Their size-dependent photophysical properties still require considerable attention.

So far considerable effort has been dedicated to the synthesis of various functionalized and non-functionalized C-NanoD based on thermal oxidation of suitable molecular precursors [14], [15], combustion of carbon soot [16] or natural gas [17], laser ablation or electrochemical oxidation of graphite [18], [19], microwave-heating of sucrose [20], [21], and thermal degradation of polysaccharides [22], [23]. The fundamental studies and applications of C-NanoD have also aroused intensive interest. Although great effort has been made in improving the quantum yield (ΦS) and surface functionalization of C-NanoD, not much focus is spent on the analytical separation of fluorescent C-NanoD. We suspect that most C-NanoD samples synthesized to date are in fact mixtures containing various core sizes, shapes and surface-functionalized moieties of C-NanoD species; in other words, an as-synthesized C-NanoD product only represents the summation or average properties of its individual C-NanoD species. In theory, each unique C-NanoD species should bear its own distinct chemical and physical properties which require more precise and accurate studies. To our knowledge, there are only few reports on the separation of C-NanoD. Liu et al. [16] fractionated C-NanoD by gel electrophoresis. C-NanoD samples derived from soot were analyzed by capillary electrophoresis (CE) [13] and anion-exchange ion chromatography (AEIC) [24], [25]. Gel electrophoresis does not provide high separation efficiency. Although CE can enjoy excellent separation, the separated and collected fractions are minuscule which make further characterization difficult. The AEIC approach has an immediate impact on the analysis and fractionation of various other nanomaterials; however, it requires relatively expensive ion-exchange column and ammonium acetate or carbonate as the eluent. The separation largely depends on the pH of eluent and the search of the optimal separating condition is time-consuming. In addition, AEIC only separates charged components but not the neutral C-NanoD entities. The buffers for eluting the samples have to be removed from the collected fractions prior to mass spectral analysis and other characterizations. As such, there is a need to develop other better analytical separation techniques to fractionate C-NanoD.

Herein, we firstly report a reversed-phase high-performance liquid chromatographic (RP-HPLC) method for efficient separation and isolation of amino/hydroxyl-functionalized fluorescent C-NanoD fractions from an as-synthesized C-NanoD product. More importantly, RP-HPLC allows for the collection of C-NanoD fractions with scale-up capabilities. It is relatively easy to remove the solvents from the collected HPLC fractions for further characterizations. Baker and Baker [2] proposed that there is a demand for developing better synthetic routes and more detailed fundamental studies of C-NanoD properties. By collecting the separated fractions, the UV–vis absorption, photoluminescence (PL), matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF MS) and transmission electron microscopic (TEM) images of each C-NanoD fraction can be realized. It is well known that C-NanoD possesses the size-dependent photophysical properties [26]. An understanding of the chromatographic separation of C-NanoD will not only help establish composition-synthesis correlations of as-synthesized C-NanoD but will also provide useful information for photophysical and chemical properties of each C-NanoD fraction. This information can provide important insights in catalysis and nanoscale electronic device research. Our developed RP-HPLC technology could open up new initiatives on extensive studies of individual C-NanoD species in the biomedical, catalysis, electronic and optical device, energy storage, material, and sensing fields. It can also provide a methodology to select and harvest the most fluorescent C-NanoD fractions for bioimaging, biosensing and optoelectronics device applications.

Section snippets

Chemicals and reagents

Chitosan (85%, 100–300 kDa) was acquired from Chengdu Kelong Chemical Reagent Factory (Chengdu, China). Glacial acetic acid was obtained from Chengdu Chemical Plant (Chengdu, China). HPLC-grade methanol (MeOH) was purchased from Labscan (Bangkok, Thailand). 2,5-Dihydroxybenzoic acid (DHB, 98%) was from Sigma (St. Louis, MO, USA). Water was purified through a Milli-Q-RO4 water purification system (Millipore, Bedford, MA, USA) with a resistivity higher than 18  cm. All reagents of analytical

Characterization of C-NanoD product

C-NanoD has been employed for bioimaging in various live cells because of their bright and stable fluorescence, good water-solubility, biocompatibility, and nontoxicity [3], [4], [5]. Water-soluble amino/hydroxyl-functionalized fluorescent C-NanoD sample was synthesized by carbonizing a mixture of chitosan and glacial acetic acid at a mild temperature of 180 °C with ΦS of 7.20%. Fig. 1A displays the UV–vis absorption spectra of the reagents chitosan/acetic acid ((a) red line) and the as-prepared

Conclusion

We have developed a RP-HPLC methodology to fractionate and study a complex mixture of the as-prepared amino/hydroxyl-functionalized fluorescent C-NanoD product. The separation coupled with UV–vis absorption, fluorescence, MS and TEM proves to be extremely useful in identifying the core size, spectral feature, characteristics and structural features of C-NanoD fractions present in the sample. The elution order follows approximately with their core sizes from the smallest to the largest. The

Acknowledgments

Financial supports from the Hundred Talent Programme of Shanxi Province, HKBU Faculty Research Grant (FRG1/13–14/039) and National Natural Science Foundation of China (21175086) are gratefully acknowledged. We would express our sincere thanks to Ms. Winnie Y.K. Wu of the Institute of Advanced Materials for taking the TEM images and Ms Silva T. Mo of the Department of Chemistry, Hong Kong Baptist University for acquiring the MALDI-TOF MS. The TEM was supported by the Special Equipment Grant from

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  • Cited by (0)

    1

    Exchange students.

    2

    Postdoctoral research fellow on visit to Hong Kong Baptist University.

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